Enrichment of histone H5-synthesizing polysomes by indirect immunoadsorption. Purification of H5 mRNA.
A method of purifying H5 mRNA from immature hen erythrocytes is described. Polysomes from red blood cells of anemic animals were allowed to react with affinity-chromatography-purified rabbit anti-H5 antibodies and immunoadsorbed to cellulose-insolubilized sheep and anti-rabbit antibodies. This method produced a 10- to 15-fold enrichment of the H5 mRNA, which was further purified by sucrose gradient centrifugation, poly(U)-Sepharose chromatography, and size fractionation in denaturing polyacrylamide gels. The H5 mRNA activity was essentially pure, as shown by translation in a reticulocyte cell-free system and immunoprecipitation with H5-antibodies. The proportion of H5 mRNA has been estimated to lie in the range 0.4-0.6% of all cellular mRNAs. These values are in agreement with the estimated relative synthesis of H5 in the immature erythrocyte.
D000818:Animals; D000936:Antigen-Antibody Complex; D002645:Chickens; D002846:Chromatography, Affinity; D004912:Erythrocytes / Q000378:metabolism; D005260:Female; D006657:Histones / Q000235:genetics*; D007106:Immune Sera; D005779:Immunodiffusion; D011132:Polyribosomes / Q000378:metabolism*; D014176:Protein Biosynthesis; D012333:RNA, Messenger / Q000235:genetics* / Q000302:isolation & purification
D000936:Antigen-Antibody Complex; D006657:Histones; D007106:Immune Sera; D012333:RNA, Messenger